Name: Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation (Video) | JoVE Journal
Uploaded: 2015-04-29T14:00:00.000+00:00
Duration: 8 min 21 s
Description: Learn to isolate murine peritoneal macrophages for gene expression analysis upon Toll-like receptor stimulation. This protocol uses thioglycolate medium to enhance yield and enables immunology research.

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5 minutes

This work was supported by a grant from the&#160;Natural Sciences and Engineering Research Council of Canada (NSERC, grant no 298515-2011).&#160;AL is the recipient of a Ph.D. scholarship from the Natural Sciences and Engineering Research Council of Canada (NSERC),&#160;and MS was supported from a grant from the Canadian Institutes of Health Research (CIHR, grant no. MOP123246).

All procedures were performed in accordance with the Canadian Council on Animal Care guidelines after approval by the institutional Animal Care Committee of the Centre de recherche du Centre Hospitalier de l&#8217;Universit&#233; de Montr&#233;al (CRCHUM).
1. Isolation, Identification, and Culture of Murine Peritoneal Macrophages
<ol>
	<li>Prepare 3.8% brewer&#8217;s thioglycollate medium. To do so, suspend 38 g of thioglycollate medium in 1,000 ml of distilled water. Bring solution to boil to dissolve the medium completely. Sterilize by autoclaving at 121 &#176;C for 15 min. Store up to 3 months in the dark, at RT18. 
	&#8203;NOTE: Discard if turbidity develops which indicates a bacterial contamination. The solution can be kept for up to one year in the dark if kept sterile.</li>
	<li>Using 1 ml syringes attached to 23 G needles, inject 1 ml of 3.8% Brewer thioglycollate medium into the peritoneal cavity of each mouse and wait for 3 days. Use new syringe and needle for each mouse.</li>
	<li>Anesthetize mice by intraperitoneal injection of sodium pentobarbital (100 mg/kg) and euthanize mice by cervical dislocation. Confirm proper anesthetization by checking the respiratory rate. Usually rapid respirations indicate that the mouse is not deeply anaesthetized.</li>
	<li>Wash the abdomen of each mouse with 70% ethanol. Using a scissor, perform a lateral incision along the bottom midline of the peritoneum.</li>
	<li>Using a forceps, pull back the abdominal skin to expose the transparent peritoneal skin. Using 5 ml syringes attached to 20 G needles, inject 5 ml of cold DPBS into the peritoneal cavity of each mouse.</li>
	<li>Perform a gentle massage on the peritoneal cavity and then aspirate the fluid carefully without puncturing any organ. Remove the needle and dispense the peritoneal fluid into 50 ml conical centrifuge tubes.</li>
	<li>Centrifuge for 10 min at 400 x g in a refrigerated centrifuge. Cells must stay cold during the whole procedure. Discard supernatant and resuspend cell pellet in RPMI medium 1640.</li>
	<li>Using a hemocytometer, count cells&#160;and adjust to cell density to 1 x 106&#160;cells/ml.&#160;</li>
	<li>Characterize the phenotype of isolated cells by flow cytometry using 1 x 106 cells per mouse and antibodie against F4/80 (a surface antigen&#160;expressed on macrophages).</li>
</ol>
2. Cell Treatments
<ol>
	<li>Directly, after isolation, add 1 x 106&#160;cells into each well. Leave the murine peritoneal macrophages to adhere into the 6-well plates by culturing them for 1 to 2 hr at 37 &#176;C. Remove non-adherent cells by gently washing 3 times with warm PBS.</li>
	<li>Subsequently, culture cells in 900 &#181;l serum-free DMEM for 24 hr in the presence of the following TLR ligands: (Pam3CSK4-TLR1/2 (0.5 mg/ml); Poly(I:C)-TLR3 (10 mg/ml); LPS-TLR4 (100 ng/ml); Flagellin-TLR5 (100 ng/ml); FSL1-TLR6/2 (100 ng/ml); ssRNA40-TLR7 (1 &#181;g/ml); ODN1826-TLR9 (1 &#181;M). 
	NOTE: Prepare for each ligand a 10x stock solution. Add 100 &#181;l of the latter to each well, thus performing a 10-fold dilution.</li>
</ol>
3. RNA Isolation
<ol>
	<li>Remove all medium and lyse cells directly in the six-well plates by adding 1 ml TRIzol to each well and passing the cell lysate several times through a pipette. Incubate the homogenised samples for 5 to 10 min at RT, to allow the complete dissociation of nucleoprotein complexes.</li>
	<li>Transfer lysate into 1.5 ml RNase- and DNase-free microcentrifuge tubes. Add 0.2 ml chloroform per 1ml TRIzol. Shake tubes vigorously by hand for 15 sec and incubate them at RT for 5 to 10 min. Centrifuge at 12,000 x g for 15 min at 4 &#176;C. Observe the mixture separating into a lower red phase (phenol-chloroform), an interphase and a colorless upper aqueous phase. RNA remains exclusively in the aqueous phase.</li>
	<li>Transfer carefully the upper aqueous phase to a fresh tube without disturbing the interphase. Precipitate the RNA from it by mixing with 0.5 ml of isopropyl alcohol. Incubate samples at RT for 10 min and centrifuge at 12,000 x g for 10 min at 4 &#176;C. The RNA precipitates forming a white pellet on the side and bottom of the tube.</li>
	<li>Remove supernatant. Wash the RNA pellet once with 1 ml of 75% ethanol. Mix the samples by vortexing and centrifuge at 7,500 x g for 5 min at 4 &#176;C. Briefly dry the RNA (air dry for 10 min). Dissolve RNA in 0.1 ml DEPC (RNAse free water) and incubate for 10 min at 55 &#176;C.</li>
	<li>Quantify RNA using a spectrophotometer. To do so, dilute samples 100 times in DEPC water and read at wavelengths of 260 nm. The RNA concentration will then be: OD260 x 40 ng/ul x dilution factor.</li>
	<li>Equalize RNA concentrations and synthesize&#160;cDNA as per manufacturer&#8217;s instructions using RT-PCR System for First-Strand cDNA Synthesis Kit. As per manufacturer&#8217;s instructions, measure gene mRNA levels by real-time PCR (45 cycles) in a Real-Time DNA detection system. Normalize gene expression levels with two housekeeping gene for example &#946;-actin and GAPDH. 
	NOTE: Normalize RNA concentration to 500 &#181;g/ml and use 0.5 &#181;g for cDNA synthesis.</li>
</ol>

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<li>Nemeth, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hepcidin+regulates+cellular+iron+efflux+by+binding+to+ferroportin+and+inducing+its+internalization%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization.</a> Science. 306 (5704), 2090-2093 (2004).</li>
<li>Constante, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Distinct+requirements+for+Hfe+in+basal+and+induced+hepcidin+levels+in+iron+overload+and+inflammation%5BTitle%5D)+AND+%22Am+J+Physiol+Gastrointest+Liver+Physiol%22%5BJournal%5D)">Distinct requirements for Hfe in basal and induced hepcidin levels in iron overload and inflammation.</a> Am J Physiol Gastrointest Liver Physiol. 291 (2), G229-G237 (2006).</li>
<li>Zhang, X., Goncalves, R., Mosser, D. M. The isolation and characterization of murine macrophages. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aZhang%2C+X+%22Curr+Protoc+Immunol%22">Curr Protoc Immunol</a>. Chapter 14 (Unit 14 11), (2008).</li>
<li>Ray, A., Dittel, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+mouse+peritoneal+cavity+cells%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Isolation of mouse peritoneal cavity cells.</a> J Vis Exp. (35), (2010).</li>
<li>Leijh, P. C., van Zwet, T. L., ter Kuile, M. N., van Furth, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+thioglycolate+on+phagocytic+and+microbicidal+activities+of+peritoneal+macrophages%5BTitle%5D)+AND+%22Infect+Immun%22%5BJournal%5D)">Effect of thioglycolate on phagocytic and microbicidal activities of peritoneal macrophages.</a> Infect Immun. 46 (2), 448-452 (1984).</li>
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<li>Li, Y. M., Baviello, G., Vlassara, H., Mitsuhashi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Glycation+products+in+aged+thioglycollate+medium+enhance+the+elicitation+of+peritoneal+macrophages%5BTitle%5D)+AND+%22J+Immunol+Methods%22%5BJournal%5D)">Glycation products in aged thioglycollate medium enhance the elicitation of peritoneal macrophages.</a> J Immunol Methods. 201 (2), 183-188 (1997).</li>
<li>Layoun, A., Santos, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bacterial+cell+wall+constituents+induce+hepcidin+expression+in+macrophages+through+MyD88+signaling%5BTitle%5D)+AND+%22Inflammation%22%5BJournal%5D)">Bacterial cell wall constituents induce hepcidin expression in macrophages through MyD88 signaling.</a> Inflammation. 35 (4), 1500-1506 (2012).</li>
<li>Oppenheim, J. J., Yang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Alarmins%3A+chemotactic+activators+of+immune+responses%5BTitle%5D)+AND+%22Curr+Opin+Immunol%22%5BJournal%5D)">Alarmins: chemotactic activators of immune responses.</a> Curr Opin Immunol. 17 (4), 359-365 (2005).</li>
<li>Akira, S., Uematsu, S., Takeuchi, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pathogen+recognition+and+innate+immunity%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Pathogen recognition and innate immunity.</a> Cell. 124 (4), 783-801 (2006).</li>
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</ol>

The authors have no competing financial interests.

Macrophages are crucial for survival and provide a tempting target to manipulate the host for immunological objectives. The discovery of TLRs and other recognition molecules have conducted the macrophages to the centre of immunological debate. Macrophages respond to a variety of stimuli, including cytokines, damage-associated molecular pattern molecules (DAMPs)20 and molecules associated with groups of pathogens (PAMPs)21. These different stimuli responses represent the course of macrophages activat...

The reticuloendothelial phagocytic system is composed of cells in various tissues and organs such as bone marrow, blood, liver and spleen. Macrophages are extensively distributed around the body, where they notably participate in innate and adaptive immune responses to control and clear infections. In addition to their role in host defense, macrophages also play an important role in wound healing and in maintaining tissue homeostasis1,2. Furthermore, macrophages are not only important to immune function but also actively participate in iron homeostasis3. In the body, approximately 80% of iron is present in hemoglobin within erythrocytes, which when senescent are phagocytosed by macrophages4. Daily, these macrophages recycle 25 mg of erythrocyte-derived iron and provide its transport into the plasma5. Moreover, during infection and inflammation, pro-inflammatory macrophages sequester serum iron to reduce iron availability to pathogens, at both the systemic and local levels6-8. As well, studies have shown that macrophages and mainly hepatocytes produce an antimicrobial peptide named hepcidin that is considered the master&#160;regulator of&#160;iron&#160;metabolism9,10. Hepcidin is mainly increased by inflammatory stimuli and is partially responsible for iron sequestration in macrophage upon chronic inflammation11-13.&#160;As hepcidin expression in macrophages is not very well understood, we studied the possible role of Toll-like receptors (TLRs) in this regulation. The TLRs&#160;are primarily found on&#160;macrophages and play a central role in&#160;their activation. In addition, LPS induced hepcidin expression in the liver is dependent on TLR413. Therefore, to execute our study, we used a method based on the isolation of murine peritoneal macrophages.
Macrophage cell lines are broadly used in macrophage studies; nonetheless extended culture can provoke gene loss and decreased immune functions in these cell lines. Thus, isolation of macrophages from peritoneal cavity is crucial.
The mouse peritoneal cavity presents an ideal site to harvest macrophages13-15. Isolated murine peritoneal macrophages are convenient for several studies regarding their immunological function. However, the number of macrophages in the peritoneum is insufficient for extensive studies and is estimated around 1 x 106 macrophages per mouse. Thus, to raise macrophage output, a sterile eliciting agent such as thioglycollate was injected into the peritoneal cavity preceding the cell harvest. After thioglycollate injection, the yield of macrophages per mouse was increased by 10-fold. Despite the increase in macrophages yield, Brewer&#8217;s thioglycollate medium acts as an irritant that induces an inflammatory response, resulting in the recruitment of macrophages, which may but not necessary affect gene expression. Hence, a control group consisting of non-treated macrophages must be included in each experiment. In our hands, hepcidin expression which is highly stimulated by inflammation was not detected in non-treated thioglycollate elicited peritoneal macrophages. Moreover, studies have shown that Brewer&#8217;s thioglycollate recruits numerous macrophages, but does not activate them16. On the other hand, Brewer&#8217;s thioglycollate elicited macrophages showed an increase in lysosomal enzyme but a decrease in&#160;killing ingested microorganisms17. However, the phagocytic capacity was not affected when compared with non-elicited macrophages16.
Once cultured in dishes, the peritoneal macrophages become adherent, therefore allowing their separation from other type of cells isolated from the peritoneal cavity. Subsequently, the isolated macrophages were challenged with different TLRs agonists. Finally, mRNA was extracted&#160;from the cultured&#160;cells and gene expression was analysed using quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR).

<table><tbody><tr><td>C57BL/6 mice</td><td>Charles River Laboratories, Inc. (Wilmington, MA, USA)</td><td>475</td><td></td></tr><tr><td>Thioglycollate</td><td>Sigma-Aldrich, (St. Louis, MO)</td><td>19032-500G</td><td></td></tr><tr><td>70% ethanol</td><td></td><td></td><td></td></tr><tr><td>10% sodium pentobarbital (Used for mice anesthesia (80 mg/kg, i.p.))</td><td></td><td></td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s phosphate-buffered saline (DPBS), placed on ice and will serve to harvest macrophages</td><td>WISENT INC Canada (QC)</td><td>311-425-CL</td><td></td></tr><tr><td>RPMI medium 1640 (Supplement with penicillin, streptomycin, L-glutamine, and 10 % fetal calf serum).</td><td>WISENT INC Canada (QC)</td><td>350-000-CL</td><td></td></tr><tr><td>1 and 5 ml syringes</td><td>BD USA (NJ)</td><td>309659</td><td></td></tr><tr><td>6-well plates</td><td>Corning Incorporated (NY, USA)</td><td>MCT-150-C</td><td></td></tr><tr><td>Bacterial lipoprotein Pam3CSK4 (0.5 mg/ml)</td><td>InvivoGen (San Diego, USA)</td><td>TLRL-pm25</td><td></td></tr><tr><td>Polyionosine&amp;ndash;polycytidylic acid (Poly(I:C)) (10 mg/ml)</td><td>InvivoGen (San Diego, USA)</td><td>TLRL-PIC</td><td></td></tr><tr><td>LPS from Escherichia coli 055:B5 (100 ng/ml)</td><td>InvivoGen (San Diego, USA)</td><td>L2880</td><td></td></tr><tr><td>Purified flagellin from Salmonella typhimurium (100 ng/ml)</td><td>InvivoGen (San Diego, USA)</td><td>TLR-FLIC-10</td><td></td></tr><tr><td>Lipoprotein synthetic FSL1 (100 ng/ml)</td><td>InvivoGen (San Diego, USA)</td><td>TLR-FSL</td><td></td></tr><tr><td>ssRNA derived from the HIV-1 long terminal repeat ssRNA40 (1 &amp;mu;g/ml)</td><td>InvivoGen (San Diego, USA)</td><td>TLR-LRNA-40</td><td></td></tr><tr><td>Type B CpG oligonucleotide ODN1826 (1 &amp;mu;M)</td><td>InvivoGen (San Diego, USA)</td><td>11B16-MM</td><td></td></tr><tr><td>TRIzol</td><td>Invitrogen, (Burlington, ON, Canada)</td><td>15596-026</td><td></td></tr><tr><td>20 G and 23 G needles</td><td>BD USA (NJ)</td><td>305175</td><td></td></tr><tr><td>Scissor</td><td></td><td></td><td></td></tr><tr><td>Forceps</td><td></td><td></td><td></td></tr><tr><td>50 ml conical tubes placed on ice</td><td>Sarstedt (Newton, MA, USA)</td><td>62.547.205</td><td></td></tr><tr><td>Red Blood Cells Lysis Buffer</td><td>Sigma-Aldrich, (St. Louis, MO)</td><td>R7757-100ML</td><td></td></tr><tr><td>Refrigerated centrifuge</td><td></td><td></td><td></td></tr><tr><td>Hemocytometer</td><td></td><td></td><td></td></tr><tr><td>F4/80 antibody</td><td>BIO-RAD ( CA, USA)</td><td>MCA497APC</td><td></td></tr><tr><td>CD16/CD32 antibodies</td><td>Pharmingen, {Mississauga, ON, CA)</td><td>553141</td><td></td></tr><tr><td>Flow cytometer</td><td>Coulter Epics Elite counter, Coulter, (Hialeah, FL,USA)</td><td></td><td></td></tr><tr><td>1.5 ml Eppendorf tubes</td><td>Axygen Scietific (CA,USA)</td><td>3516</td><td></td></tr><tr><td>Chloroform</td><td>Fisher Scientific (ON, Canada)</td><td>UN1888</td><td></td></tr><tr><td>Isopropyl alcohol</td><td>JT Baker (PA, USA)</td><td>70566</td><td></td></tr><tr><td>75% ethanol (in DEPC treated water)</td><td>Commercial Alchohols (QC, Canada)</td><td>17394</td><td></td></tr><tr><td>0.01% diethyl pyrocarbonate (DEPC) treated water (let stand overnight and autoclave)</td><td>Sigma-Aldrich, (St. Louis, MO)</td><td>216.542.8</td><td></td></tr><tr><td>Omniscript RT-PCR system</td><td>Qiagen, (Mississauga, ON, Canada)</td><td>205113</td><td></td></tr><tr><td>Rotor Gene 3000</td><td>Montreal Biotech, (Kirkland, QC, Canada)</td><td></td><td></td></tr><tr><td>QuantiTect SYBR Green I PCR kits</td><td>Qiagen, (Mississauga, ON, Canada)</td><td>204141</td><td></td></tr></tbody></table>

isolation of murine peritoneal macrophages to carry out gene expression analysis upon toll-like receptors stimulation

Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. Macrophages express surface Toll-like receptors (TLRs), which recognize molecular patterns conserved through evolution in a wide range of microorganisms. TLRs play a central role in macrophage activation which is usually associated with gene expression alteration. Macrophages are critical in many diseases and have emerged as attractive targets for therapy. In the following protocol, we describe a procedure to isolate murine peritoneal macrophages using Brewer&#8217;s thioglycollate medium. The latter will boost monocyte migration into the peritoneum, accordingly this will raise macrophage yield by 10-fold. Several studies have been carried out using bone marrow, spleen or peritoneal derived macrophages. However, peritoneal macrophages were shown to be more mature upon isolation and are more stable in their functionality and phenotype. Thus, macrophages isolated from murine peritoneal cavity present an important cell population that can serve in different immunological and metabolic studies. Once isolated, macrophages were stimulated with different TLR ligands and consequently&#160;gene expression was evaluated.

We first characterized the isolated murine peritoneal macrophages by flow cytometry. To do so, we used (F4/80) antibodies that specifically recognize markers only expressed by macrophages. This characterization is required to determine the percentage of isolated macrophage and to distinguish them among cells obtained during the isolation process. As shown in (Figure 1), the percentage of cells expressing the antigen F4/80 was consistently found to be above 95%. Next, to study gene expression in macrophag...

We describe here a simple protocol to isolate murine peritoneal macrophages. This procedure is followed by RNA extraction to carry out gene expression analysis upon Toll-like receptors stimulation.

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. ...

The overall goal of this procedure is to isolate murn peritoneal macrophages and to perform gene expression analysis upon the stimulation of toll-like receptors. This is accomplished by first injecting. 3.8%of brewer thi glyco medium into the peritoneal cavity of the mouse to increase monocyte migration into the peritoneum and therefore increase macrophage yield after three days, euthanize the mouse and inject cold ECCOs phosphate buffer saline into the peritoneal cavity to harvest the cells.Following a gentle massage on the peritoneal cavity, the peritoneal fluid is carefully aspirated. Next, the obtained cells are cultured and the elicited peritoneal macrophages are characterized by flow cytometry. The final step is to stimulate these macrophages with different toll-like receptor or TLR ligands and isolate the RNA from the samples.Ultimately, quantitative reverse transcriptase polymerase chain reaction is used to measure expression of the gene of interest. The goal of the following procedure is to isolate, mirroring peritoneal microphages using brewer's TIO glycol medium. This medium will increase monocyte migration into the peritoneum and therefore increase microphage yield.Once isolated microphages will be stimulated with different toll-like receptor ligands to study the molecular regulation of hepcidin as our gene of Pinterest. To begin, prepare 3.8%brewer's bio glyco eight medium as described in the text protocol using one milliliter syringes attached to 23 gauge needles. Inject one milliliter of 3.8%brewer's bio glycol eight medium into the peritoneal cavity of each mouse, and wait for three days.Use a new syringe and needle for each mouse following mouse anesthetization and euthanasia as described in the text protocol, soak the abdomen of each mouse with 70%ethanol. Perform a lateral incision along the bottom midline of the peritoneum with scissors. Use forceps to pull back the abdominal skin and expose the transparent peritoneal skin.Then inject five milliliters of cold ECCOs phosphate buffered saline into the peritoneal cavity of each mouse with five milliliter syringes attached to 20 gauge needles. Perform a gentle massage on the peritoneal cavity and then aspirate the fluid carefully without puncturing any organ. Remove the needle and dispense the peritoneal fluid into 50 milliliter conical centrifuge tubes.Next centrifuge the collected peritoneal fluid for 10 minutes at 400 times G or 1500 RPM in a refrigerated centrifuge, cells should be kept cold throughout the procedure. Discard the supernatant and resuspend the cell pellet in RPMI 1640 medium. Using a hemo cytometer, count the cells and adjust the samples to a cell density of 1 million cells per milliliter.Characterize the phenotype of isolated cells by flow cytometry to confirm macrophage enrichment. Using 1 million cells per mouse and antibodies against F four 80, which is a surface antigen expressed on macrophages directly. After isolation, add 1 million cells into each well of a six.Well plate. Leave the murine peritoneal macrophages to adhere to the wells by culturing them for one to two hours at 37 degrees Celsius. Following incubation, remove non-adherent cells by gently watching three times with warm phosphate buffered saline.Then add 900 microliters of serum free DMEM to the cells. Prepare 10 x stock solutions of each toll-like receptor or TLR ligand as listed in the text protocol. Then add 100 microliters of the stock solutions to each well, thus performing a tenfold dilution culture.The cells for 24 hours in the presence of the TLR ligands. To perform the RNA isolation first, remove all medium and lyce the cells directly in the six well plates. By adding one milliliter of triol reagent to each well and passing the cell lysate several times through a pipette.Then incubate the homogenized samples for five to 10 minutes at room temperature to permit the complete dissociation of nuclear protein complexes. Following incubation, transfer the lysate into 1.5 milliliter RNAs and DNA spree micro centrifuge tubes, and add 0.2 milliliters of chloroform per one milliliter of triol reagent. Shake the tubes vigorously by hand for 15 seconds before incubating them at room temperature for five to 10 minutes.Then centrifuge at 12, 000 times G for 15 minutes at four degrees Celsius. Observe the mixture separating into a lower red phenol chloroform phase, an interphase and a colorless upper aqueous phase. RNA remains exclusively in the aqueous phase.Carefully transfer the upper aqueous phase to a fresh tube without disturbing the interphase phase. Precipitate the RNA from it by mixing with 0.5 milliliters of isopropyl alcohol after incubating the samples at room temperature for 10 minutes. Centrifuge at 12, 000 times G for 10 minutes at four degrees Celsius.The RNA precipitates forming a white pellet on the side and bottom of the tube. Remove the supernatant and wash the RNA pellet once with one milliliter of 75%ethanol. Mix the samples by vortexing and then centrifuge at 7, 500 times G for five minutes at four degrees Celsius.After removing the supernatant, briefly air dry the RNA for 10 minutes. Then dissolve the RNA in 100 microliters of RNA free water and incubate for 10 minutes at 55 degrees Celsius. Next, quantify the RNA using a spectrophotometer to do so.Dilute samples 100 times in RNA free water and read at wavelengths of 260 nanometers equalize RNA concentrations and synthesize CD NA as per manufacturer's instructions. Using the R-T-P-C-R system for first strand CD NA synthesis kit. Then measure gene Mr.NA levels by realtime PCR in a real-time, DNA detection system, normalizing gene expression levels with two housekeeping genes.The isolated mirroring peritoneal macrophages were characterized by flow cytometry to determine the percentage of isolated macrophage and to distinguish them among cells obtained during the isolation process. The percentage of cells expressing the antigen F four 80 was consistently found to be above 95%The isolated cells were then treated with several TLR ligands and mRNA. Levels of hepcidin were measured by R-T-P-C-R tolike receptor one and two, tolike receptor four.An olic receptor six and two ligands were capable of stimulating hepcidin mRNA in mirroring macrophages. Together these results demonstrate the usefulness of this protocol to successfully isolate, mirroring peritoneal macrophages and to precisely investigate the molecular regulation of gene expression. After watching this video, you will have a good understanding of how to isolate, identify, and culture mirroring peritoneal macrophages.

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Learn to isolate murine peritoneal macrophages for gene expression analysis upon Toll-like receptor stimulation. This protocol uses thioglycolate medium to enhance yield and enables immunology research.

Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

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